Lab Methods 2011

Lab Methods 2011 - Lab Methods to Assess Immune Competence...

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Lab Methods to Assess Immune Competence Innate Immunity Enumeration of PMNs PMNs by flow (CD18, CD11)) NKs by flow (CD16) NBT dye reduction test T Cell Mediated Immunity Enumeration of T cells CD4, CD8 lymphocyte response to mitogens and/or antigens T cell subsets by flow cytometry (CD3, CD4, CD8, CD25, CD45R0,CD45RA) DTH anergy testing B Cell Mediated Enumeration of B cells B cells by flow (CD19, CD20) Serum Ig classes and subclasses by nephelometry Antibody response to specific proteins and polysaccharide antigens (Abs to tetanus/diphtheria toxoids; Abs to PRP H. influenza type B vaccine) Lab Methods to Assess Infection •ELISA •Western Immunoblot Lab Methods to Assess Type I and Type IV Allergy Skin reactions RAST 1 Complement Activity CH50
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I. Enumeration of Cell Types in Whole Blood 2
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Figure 1. PMA-stimulated NBT reduction of neutrophils from a normal adult individual. Nearly 100% of all normal neutrophils gave NBT reduction. The positive cells were transformed, enlarged, and had lost their nuclear lobulation and reduced NBT to formazan deposits. Table 1. Estimation of NBT reduction test by counting all the polymorphonuclear leukocytes containing formazan deposit. Studied individuals No. tested No. stimulated PMN CGD patients 9 <10 Carriers of CGD 7 45 Cord blood Normal controls 16 50 98 100 Figure 2. PMA stimulated NBT reduction of neutrophils from a CGD patient. Neutrophils show no NBT reduction. These cells were not transformed and failed to reduce NBT. Figure 3. PMA-stimulated NBT reduction of neutrophils from a heterozygous carrier of CGD. This shows the two cell populations of a) unaffected polymorphs showing transformation with black formazan deposition and b) affected polymorphs showing Assessment of Innate Immunity: ROS and neutrophils 4
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T Cell Mediated Immunity Enumeration of T cells CD4, CD8 lymphocyte response to mitogens and/or antigens T cell subsets by flow cytometry DTH anergy testing 5
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Figure A-23 Isolation of WBC populations 6
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II. Antigen or mitogen stimulated lymphocyte proliferation as a measure of CMI: Recall antigens : tetanus toxoid, C. albicans antigen and streptokinase 7
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Definition: Flow Cytometry is the technological process that allows for the individual measurements of cell fluorescence and light scattering. This process is performed at rates of thousands of cells per second. This information can be used to individually sort or separate subpopulations of cells. III. Measures of Cell Types based on Intrinsic and Extrinsic characteristics using Flow Cytometry Applications: Immunofluorescence Cell Cycle Kinetics Cell Kinetics Genetics Molecular Biology Animal Husbandry (and Human as well) Microbiology Biological Oceanography Parasitology Bioterrorism Clinical Applications: •Origin of leukemias and lymphoma to
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This note was uploaded on 10/08/2011 for the course PHAR 4030 taught by Professor Dr.miller during the Spring '11 term at UConn.

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Lab Methods 2011 - Lab Methods to Assess Immune Competence...

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